Application of nilotinib in preparation of medicine for treating diabetic nephropathy

Abstract

The invention relates to the technical field of medicines. The invention provides application of nilotinib in preparation of a medicine for treating diabetic nephropathy and a medicine for treating podocyte injury. According to the nilotinib disclosed by the invention, through inhibiting ZAK alpha phosphorylation, the expression of P38 phosphorylation and JNK phosphorylation can be down-regulated, the ratio level of urine microalbumin / creatinine is remarkably reduced, the number of WT1 positive cells is remarkably increased, DKD podocyte damage is relieved, and the effect of treating diabetic nephropathy is achieved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of nilotinib in preparing a drug for treating diabetic nephropathy. Background Art

[0002] Diabetic Kidney Disease (DKD) is the main cause of end-stage renal disease and directly affects the recovery and quality of life of diabetic patients. As terminally differentiated cells on the outside of the glomerular basement membrane, podocyte damage is an important cause of DKD, and the degree of damage is closely related to the severity of the disease. More and more studies have shown that in addition to hyperglycemia, there are other key mechanisms that dominate or synergistically participate in the progression of DKD. Current treatments for DKD include traditional RAS inhibitors, SGLT2 inhibitors, and new non-steroidal selective mineralocorticoid receptor antagonists, but the therapeutic effect is limited.

[0003] Podocytes are highly sensitive to injury stress. Ribosomes are important sites for protein synthesis. When they are severely damaged, they initiate stress responses to regulate cell fate. Ribosome stress responses are mediated by the autophosphorylation of ZAKα to initiate the P38-MAPK-mediated apoptosis pathway, which participates in regulating cell damage. Literature has reported that the activation of P38 and JNK downstream of ZAKα promotes podocyte apoptosis and participates in the progression of DKD.

[0004] Based on the above, it is very necessary to explore a drug with better therapeutic effect on DKD. Summary of the invention

[0005] The purpose of the present invention is to provide the use of nilotinib in the preparation of a drug for treating diabetic nephropathy, wherein the nilotinib can significantly reduce the level of urine microalbumin / creatinine ratio, increase the number of WT1 positive cells, alleviate DKD podocyte damage, and achieve the effect of treating diabetic nephropathy.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides application of nilotinib in preparing a medicine for treating diabetic nephropathy.

[0008] Preferably, the nilotinib can treat diabetic nephropathy by inhibiting ZAKα phosphorylation, down-regulating the expression of P38 phosphorylation and JNK phosphorylation, and alleviating DKD podocyte injury.

[0009] The present invention provides application of nilotinib in preparing a drug for treating podocyte injury.

[0010] The present invention also provides a medicine for treating diabetic nephropathy, comprising nilotinib and pharmaceutically acceptable excipients.

[0011] Preferably, the dosage form of the drug includes liquid or solid dosage form.

[0012] Preferably, the pharmaceutically acceptable excipients include one or more of fillers, wetting agents, binders, disintegrants, and lubricants.

[0013] By adopting the above technical solution, the present invention has the following beneficial effects:

[0014] Nilotinib described in the technical solution of the present invention can significantly downregulate the expression of P38 phosphorylation and JNK phosphorylation, significantly reduce the level of urine microalbumin / creatinine ratio, significantly increase the number of WT1 positive cells, alleviate DKD podocyte damage, and achieve the effect of treating diabetic nephropathy by inhibiting ZAKα phosphorylation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The ratio of urinary microalbumin to creatinine in mice in different treatment groups;

[0016] Figure 2 The expression of WT1 in podocytes of mice in different treatment groups ( Figure 2 A in the figure shows the immunofluorescence expression of WT1 and Nephrin in glomeruli, where red indicates WT1 and green indicates Nephrin; B shows the semi-quantitative analysis results of WT1 in glomeruli, n=5, *P<0.05). DETAILED DESCRIPTION

[0017] The present invention provides an application of nilotinib in preparing a medicine for treating diabetic nephropathy.

[0018] In the present invention, nilotinib can inhibit ZAKα phosphorylation, down-regulate the expression of P38 phosphorylation and JNK phosphorylation, and alleviate DKD podocyte injury to treat diabetic nephropathy.

[0019] The present invention provides application of nilotinib in preparing a drug for treating podocyte injury.

[0020] The present invention also provides a medicine for treating diabetic nephropathy, comprising nilotinib and pharmaceutically acceptable excipients.

[0021] In the present invention, the dosage form of the drug includes liquid or solid dosage form.

[0022] In the present invention, the pharmaceutically acceptable excipients include one or more of fillers, wetting agents, binders, disintegrants, and lubricants.

[0023] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0024] Example 1

[0025] (I) Experimental Materials Nilotinib was purchased from TargetMol Chemicals Inc., CAS 641571-10-0. db / db mice: BKS-db / db mice, 12 weeks old, weighing 50-55 g, strain number T002407, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0026] db / m mice: 12 weeks old, weighing 20-25 g, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0027] The ELISA urine protein detection kit was purchased from Ethos Biosciences.

[0028] The creatinine test kit was purchased from BioAssay Systems with batch number DICT-500.

[0029] Reagents used for immunofluorescence: Nephrin antibody (20R-NP002) was purchased from Fitzgerald, WT1 (Wilms tumor 1) antibody (ab89901) was purchased from Abcam.

[0030] (II) Experimental process

[0031] The db / db mice were used as type I diabetic nephropathy model mice, and then the diabetic nephropathy model mice were randomly divided into two groups, namely the diabetic nephropathy control group and the diabetic nephropathy + Nilotinib treatment group; db / m mice were used as the healthy control group, with 5 mice in each group.

[0032] Diabetic nephropathy + Nilotinib treatment group (db / db+Nilotinib): db / db mice were treated with ZAKα phosphorylation inhibitor Nilotinib (10 mg / kg / d) by oral gavage for 2 weeks starting from 14 weeks of age.

[0033] Diabetic nephropathy control group (db / db): db / db mice were treated with normal saline (10 mg / kg / d) by gavage for 2 weeks starting from 14 weeks of age.

[0034] Healthy control group (db / m): no oral administration was performed.

[0035] At 16 weeks of age, mice in each group were euthanized and blood, urine and kidney samples were collected. (three)

[0037] 1. Determination of urine microalbumin to creatinine ratio (ACR)

[0038] The urine of each group of mice was collected, and the urine protein level was detected using an ELISA urine protein detection kit (see the instructions for specific operations). The reagent was equilibrated at room temperature for 1 hour, and 50 μL of standard and mouse urine samples were added to each well, and 100 μL of sample diluent was added to the negative control well. Except for the negative control well, 50 μL of horseradish peroxidase was added to each well, which was coupled with the albumin antibody and incubated at room temperature for 30 minutes; washed 10 times with sterile double distilled water (ddH2O); 100 μL of color development solution was added for 5-10 minutes; 100 μL of stop solution was added, and Varioskan was used to detect the color. TM The LUX multifunctional microplate reader detects the readings at a wavelength of 450 nm and calculates the urine albumin concentration.

[0039] Use the creatinine detection kit to determine the urine creatinine level (see the instructions for specific operations), pipette 5 μL of 50 mg / dL standard and 5 μL of mouse urine sample into a 96-well plate, add 100 μL ddH2O and 100 μL working solution (50 μL working solution A and 50 μL working solution B) to each well, and immediately use Varioskan TM The LUX multifunctional microplate reader reads the OD value at 490 nm at 0 min and the OD value at 490 nm at 5 min, and calculates the creatinine value of the sample.

[0040] Creatinine value = (OD sample 5min -OD samples 0min ) / (OD Standard 5min -OD Standard 0min )×2mg / dL.

[0041] The results are as follows Figure 1 As shown, it can be seen that the ACR level of db / db mice was significantly higher than that of db / m mice; compared with db / db mice, the ACR level of mice in the db / db+Nilotonib treatment group was significantly decreased (n=5, *P<0.05), indicating that Nilotonib treatment can significantly alleviate DKD podocyte injury.

[0042] 2. Expression of WT1 in podocytes

[0043] Immunofluorescence staining. Frozen tissue sections of mice in different treatment groups with a thickness of about 3-4 μm were taken, fixed with acetone for 7 min, and blown dry; after blocking with fetal bovine serum, primary antibodies Nephrin (1:200) and WT1 (1:100) were added and incubated overnight at room temperature; after rinsing with PBS, secondary antibodies (1:200) were added and incubated for 40 min, and PBS was rinsed three times; 4',6-diamidino-2-phenyl-indole (4',6-diamidino-2-phenyl-indole, DAPI) was used to stain the nucleus for 3 min, the slides were rinsed with tap water, dried, fixed and sealed; the slides were observed and images were taken under a fluorescence microscope.

[0044] according to Figure 2 It can be seen that the number of WT1-positive cells in the glomeruli of mice in the db / db group was significantly lower than that in the db / m group; the number of WT1-positive cells in the mice treated with db / db+Nilotonib increased significantly, indicating that Nilotonib treatment can significantly alleviate DKD podocyte injury.

[0045] In summary, it can be seen that Nilotinib described in the technical solution of the present invention can significantly downregulate the expression of P38 phosphorylation and JNK phosphorylation by inhibiting ZAKα phosphorylation, significantly reduce the level of urinary microalbumin / creatinine ratio, significantly increase the number of WT1 positive cells, alleviate DKD podocyte damage, and thus achieve the treatment of diabetic nephropathy.

[0046] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of nilotinib in the preparation of drugs for the treatment of diabetic nephropathy.

2. The use according to claim 1, characterized in that: The nilotinib can treat diabetic nephropathy by inhibiting ZAKα phosphorylation, down-regulating the expression of P38 phosphorylation and JNK phosphorylation, and relieving DKD podocyte damage.

3. Application of nilotinib in the preparation of drugs for the treatment of podocyte injury.

4. A drug for treating diabetic nephropathy, characterized in that: The invention comprises nilotinib and pharmaceutically acceptable excipients.

5. The drug according to claim 4, characterized in that The dosage form of the drug includes liquid or solid dosage form.

6. The drug according to claim 4 or 5, characterized in that The pharmaceutically acceptable excipients include one or more of fillers, wetting agents, binders, disintegrants, and lubricants.

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